Project Summary/Abstract (Project 1, Portnoy) This proposal is Project 1 within a P01 renewal, entitled ?Intracellular pathogens and innate immunity.? A central problem that we address is how intracellular pathogens are recognized by the host innate immune system and how multiple signals are integrated to induce an appropriate response, and conversely, how pathogens avoid and/or manipulate host responses to promote their pathogenesis. In Project 1, we have chosen to approach this problem by continuing a detailed analysis of Listeria monocytogenes, a facultative intracellular, food-borne pathogen that has been studied for decades as a model system with which to dissect basic aspects of infection & immunity. Previously, we concentrated on macrophage transcriptional responses to infection and learned that L. monocytogenes escapes from a phagosome and secretes a conserved and essential small signaling molecule called c-di-AMP that activates the transcription of the host type I interferon response. In collaboration with the Vance lab, we identified STING as the host cell receptor for both c-di-AMP and other c-di-nucleotides. In Aim 1, we propose to extend these studies, using bacterial mutants that either synthesize or secrete altered levels of c-di-AMP in combination with mice and host cells that have specific STING mutations that alter downstream signaling. In Aims 2 and 3, we begin to explore the role of post-transcriptional host responses including ubiquitylation of both bacterial and host proteins and the role played by autophagy. In Aim 2, we follow-up on preliminary data showing that L. monocytogenes is subject to autophagy at the phagosome, but circumvents it using two determinants, PlcA and ActA. We show that a double PlcA/ActA mutant is trapped by what appears to be LC3-associated phagocytosis (LAP). We will use bacterial and host mutants to determine if this is in fact LAP, how the bacteria avoid it, and finally, what is the role of LAP avoidance during infection and immunity. In Aim 3, we propose using mass spectrometry-based and bioinformatic tools, available in Core C, to identify the bacterial and host proteins that are ubiquitylated, and high throughput genetic screens to identify the host proteins that mediate ubiquitylation.